Cytokine biomarkers as predictive biomarkers of clinical response for glatiramer acetate

ABSTRACT

A method for treating a human subject afflicted with multiple sclerosis or a single clinical attack consistent with multiple sclerosis with a pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier, comprising the steps of determining whether the human subject is a glatiramer acetate responder by evaluating a biomarker selected from the group consisting of IL-17 concentration, TNF-α concentration, IL-2 concentration and IFN-γ concentration, or a combination thereof, in the blood of the human subject and administering the pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier to the human subject only if the human subject is identified as a glatiramer acetate responder.

This application claims benefit of U.S. Provisional Application No. 61/391,968, filed Oct. 11, 2010, the contents of which are hereby incorporated by reference into this application.

Throughout this application various publications are referenced by their full citations in parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

Multiple sclerosis (MS) is a chronic, debilitating autoimmune disease of the central nervous system (CNS) with either relapsing-remitting (RR) or progressive course leading to neurologic deterioration and disability. At time of initial diagnosis, RRMS is the most common form of the disease (1) which is characterized by unpredictable acute episodes of neurological dysfunction (relapses), followed by variable recovery and periods of clinical stability. The vast majority of RRMS patients eventually develop secondary progressive (SP) disease with or without superimposed relapses. Around 15% of patients develop a sustained deterioration of their neurological function from the beginning; this form is called primary progressive (PP) MS. Patients who have experienced a single clinical event (Clinically Isolated Syndrome or “CIS”) and who show lesion dissemination on subsequent magnetic resonance imaging (MRI) scans according to McDonald's criteria, are also considered as having relapsing MS.(2)

With a prevalence that varies considerably around the world, MS is the most common cause of chronic neurological disability in young adults.(3,4) Anderson et al. estimated that there were about 350,000 physician-diagnosed patients with MS in the United States in 1990 (approx. 140 per 100,000 population).(5) It is estimated that about 2.5 million individuals are affected worldwide.(6) In general, there has been a trend toward an increasing prevalence and incidence of MS worldwide, but the reasons for this trend are not fully understood.(5)

Current therapeutic approaches consist of i) symptomatic treatment ii) treatment of acute relapses with corticosteroids and iii) treatment aimed to modify the course of the disease. Currently approved therapies target the inflammatory processes of the disease. Most of them are considered to act as immunomodulators but their mechanisms of action have not been completely elucidated. Immunosuppressants or cytotoxic agents are also used in some patients after failure of conventional therapies. Several medications have been approved and clinically ascertained as efficacious for the treatment of RR-MS; including BETASERON®, AVONEX® and REBIF®, which are derivatives of the cytokine interferon beta (IFNB), whose mechanism of action in MS is generally attributed to its immunomodulatory effects, antagonizing pro-inflammatory reactions and inducing suppressor cells.(7) Other approved drugs for the treatment of MS include Mitoxantrone and Natalizumab.

Glatiramer Acetate

Glatiramer acetate (GA) is the active substance in Copaxone®, a marketed product indicated for reduction of the frequency of relapses in patients with RRMS. Its effectiveness in reducing relapse rate and disability accumulation in RR-MS is comparable to that of other available immunomodulating treatments.(8,9,10) Glatiramer acetate consists of the acetate salts of synthetic polypeptides containing four naturally occurring amino acids: L-glutamic acid, L-alanine, L-tyrosine and L-lysine. The average molecular weight of glatiramer acetate is between 5,000 and 9,000 Daltons. At a daily standard dose of 20 mg, GA is generally well tolerated, however response to the drug is variable. In various clinical trials, GA reduced relapse rates and progression of disability in patients with RR-MS. The therapeutic effect of GA is supported by the results of magnetic resonance imaging (MRI) findings from various clinical centers (11), however there are no validated predictive biomarkers of response to GA treatment.

A possible initial mode of action of GA is associated with binding to MHC molecules and consequent competition with various myelin antigens for their presentation to T cells.(12) A further aspect of its mode of action is the potent induction of T helper 2 (Th2) type cells that presumably can migrate to the brain and lead to in situ bystander suppression.(13) It has been shown that GA treatment in MS results in the induction of GA-specific I cells with predominant Th2 phenotype both in response to GA and cross-reactive myelin antigens. (13,14) Furthermore, the ability of GA-specific infiltrating cells to express anti-inflammatory cytokines such as IL-10 and transforming growth factor-beta (TGF-β) together with brain-derived neurotrophic factor (BDNF) seem to correlate with the therapeutic activity of GA in EAE.(15,16,17)

Clinical experience with GA consists of information obtained from completed and ongoing clinical trials and from post-marketing experience. The clinical program includes three double-blind, placebo-controlled studies in RRMS subjects treated with GA 20 mg/day.(18,19,20) A significant reduction in the number of relapses, compared with placebo, was seen. In the largest controlled study, the relapse rate was reduced by 32% from 1.98 under placebo to 1.34 under GA 20 mg. GA 20 mg has also demonstrated beneficial effects over placebo on MRI parameters relevant to RRMS. A significant effect in median cumulative number of Gd-enhancing lesions over 9 months of treatment (11 lesions in the 20 mg group compared to 17 lesions under placebo) was demonstrated.

The clinical program with GA also includes one double-blind study in chronic-progressive MS subjects,(21) one double-blind placebo-controlled study in primary progressive patients,(22) one double-blind placebo-controlled study in CIS patients(23) and numerous open-label and compassionate use studies, mostly in RRMS. The clinical use of GA has been extensively reviewed and published in the current literature (24,25,26,27).

An important tool for the therapeutic management of those with relapsing MS is the ability to determine the likelihood of treatment success. The identification of those individuals that will respond to GA has been elusive. As the therapeutic options for MS increase, the importance of being able to determine who will respond favorably to therapy and specifically to GA, has become of increasing significance.

SUMMARY OF THE INVENTION

This invention provides a method for treating a human subject afflicted with multiple sclerosis or a single clinical attack consistent with multiple sclerosis with a pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier, comprising the steps of determining whether the human subject is a glatiramer acetate responder by evaluating a biomarker selected from the group consisting of IL-17 concentration, TNF-α concentration, IL-2 concentration and IFN-γ concentration, or a combination thereof, in the blood of the human subject and administering the pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier to the human subject only if the human subject is identified as a glatiramer acetate responder.

This invention also provides a method of predicting clinical responsiveness to glatiramer acetate therapy in a human subject afflicted with multiple sclerosis or a single clinical attack consistent with multiple sclerosis, the method comprising evaluating a biomarker selected from the group consisting of IL-concentration, TNF-α concentration, IL-2 concentration and IFN-γ concentration, or a combination thereof, in the blood of the human subject, to thereby predict clinical responsiveness to glatiramer acetate.

This invention also provides a method for treating a human subject afflicted with multiple sclerosis or a single clinical attack consistent with multiple sclerosis comprising the steps of administering to the human subject a therapeutic amount of a pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier, determining whether the human subject is a glatiramer acetate responder by evaluating a biomarker selected from the group consisting of IL-17 concentration, INF-α concentration, IL-2 concentration and IFN-γ concentration, or a combination thereof, in the blood of the human subject, and continuing administration of the pharmaceutical composition if the human subject is identified as a glatiramer acetate responder, or modifying the administration of the pharmaceutical composition to the human subject if the human subject is not identified as a glatiramer acetate responder.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A: Time line showing responders' EDSS score with GA treatment.

FIG. 1B: Time line showing non-responders' EDSS score with GA treatment.

FIG. 2A: Cytokine levels secreted by the Peripheral blood mononuclear cells (PBMCs) of responders at baseline and after 2 months of GA treatment.

FIG. 2B: Cytokine levels secreted by PBMCs of non-responders at baseline and after 2 months of GA treatment.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a method for treating a human subject afflicted with multiple sclerosis or a single clinical attack consistent with multiple sclerosis with a pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier, comprising the steps of determining whether the human subject is a glatiramer acetate responder by evaluating a biomarker selected from the group consisting of IL-17 concentration, TNF-α concentration, IL-2 concentration and IFN-γ concentration, or a combination thereof, in the blood of the human subject and administering the pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier to the human subject only if the human subject is identified as a glatiramer acetate responder.

In an embodiment, administering the pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier comprises administering to the human subject three subcutaneous injections of the pharmaceutical composition over a period of seven days with at least one day between every subcutaneous injection.

In an embodiment, the pharmaceutical composition is a unit dose of a 0.5 ml aqueous solution comprising 20 mg of glatiramer acetate.

In an embodiment, the IL-17 concentration, TNF-α concentration, IL-2 concentration and IFN-γ concentration, or the combination thereof is a PBMC supernatant concentration.

In an embodiment, the IL-17 concentration, TNF-α concentration, IL-2 concentration and IFN-γ concentration, or the combination thereof is observed at pretreatment.

In an embodiment, the IL-17 concentration, INF-α concentration, IL-2 concentration and INF-γ concentration, or the combination thereof is observed at 2 months after the first administration of glatiramer acetate.

In an embodiment, if the human subject is identified as a glatiramer acetate responder, the human subject is thereafter administered the pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier as monotherapy.

In an embodiment, if the human subject is identified as a glatiramer acetate responder, the human subject is thereafter administered a pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier, and the human subject is also thereafter administered another multiple sclerosis drug which is not glatiramer acetate. In a further embodiment, the multiple sclerosis drug is selected from Interferon, Mitoxantrone and Natalizumab.

In an embodiment, if the human subject is not identified as a glatiramer acetate responder, the human subject is thereafter administered a multiple sclerosis drug which is not glatiramer acetate.

In an embodiment, if the human subject is not identified as a glatiramer acetate responder, the human subject is thereafter administered a multiple sclerosis drug which is not glatiramer acetate, and the human subject is not thereafter administered glatiramer acetate.

In an embodiment, the multiple sclerosis drug is selected from Interferon, Mitoxantrone and Natalizumab.

In an embodiment, the biomarker is IL-17 concentration.

In an embodiment, the biomarker is IL-17(A) concentration. In a further embodiment, an IL-17 concentration or an IL-17(A) concentration greater than or equal to 120 pg/ml is associated with a human subject identified as a glatiramer acetate responder.

In an embodiment, the biomarker is TNF-α concentration. In a further embodiment, a TNF-α concentration greater than or equal to 20000 pg/ml is associated with a human subject identified as a glatiramer acetate responder.

In an embodiment, the biomarker is IFN-γ concentration. In a further embodiment, an IFN-γ concentration greater than or equal to 6000 pg/ml is associated with a human subject identified as a glatiramer acetate responder.

In an embodiment, the biomarker is IL-2 concentration. In a further embodiment, an IL-2 concentration greater than or equal to 30000 pg/ml is associated with a human subject identified as a glatiramer acetate responder.

In an embodiment, the human subject is a naive patient.

In an embodiment, the human subject has been previously administered a multiple sclerosis drug other than glatiramer acetate. In a further embodiment, the previously administered multiple sclerosis drug is selected from Interferon, Mitoxantrone and Natalizumab.

This invention also provides a method of predicting clinical responsiveness to glatiramer acetate therapy in a human subject afflicted with multiple sclerosis or a single clinical attack consistent with multiple sclerosis, the method comprising evaluating a biomarker selected from the group consisting of IL-17 concentration, INF-α concentration, IL-2 concentration and IFN-γ concentration, or a combination thereof, in the blood of the human subject, to thereby predict clinical responsiveness to glatiramer acetate.

In an embodiment, the glatiramer acetate therapy comprises administering to the human subject three subcutaneous injections of a pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier over a period of seven days with at least one day between every subcutaneous injection. In a further embodiment, the pharmaceutical composition is a unit dose of a 0.5 ml aqueous solution comprising 20 mg of glatiramer acetate.

In an embodiment, the IL-17 concentration, INF-α concentration, IL-2 concentration and IFN-γ concentration, or the combination thereof is a PBMC supernatant concentration.

In an embodiment, the IL-17 concentration, INF-α concentration, IL-2 concentration and IFN-γ concentration, or the combination thereof is observed at pretreatment.

In an embodiment, the IL-17 concentration, INF-α concentration, IL-2 concentration and INF-γ concentration, or the combination thereof is observed at 2 months after the first administration of glatiramer acetate.

In an embodiment, if the human subject is identified as a glatiramer acetate responder, the human subject is thereafter administered the pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier as monotherapy.

In an embodiment, if the human subject is identified as a glatiramer acetate responder, the human subject is thereafter administered a pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier, and the human subject is also thereafter administered another multiple sclerosis drug which is not glatiramer acetate. In a further embodiment, the multiple sclerosis drug is selected from Interferon, Mitoxantrone and Natalizumab.

In an embodiment, if the human subject is not identified as a glatiramer acetate responder, the human subject is thereafter administered a multiple sclerosis drug which is not glatiramer acetate.

In an embodiment, if the human subject is not identified as a glatiramer acetate responder, the human subject is thereafter administered a multiple sclerosis drug which is not glatiramer acetate, and the human subject is not thereafter administered glatiramer acetate.

In an embodiment, the multiple sclerosis drug is selected from Interferon, Mitoxantrone and Natalizumab.

In an embodiment, the biomarker is IL-17 concentration.

In an embodiment, the biomarker is IL-17(A) concentration. In a further embodiment, an IL-17 concentration or an IL-17(A) concentration greater than or equal to 120 pg/ml is associated with a human subject identified as a glatiramer acetate responder.

In an embodiment, the biomarker is TNF-α concentration. In a further embodiment, a TNF-α concentration greater than or equal to 20000 pg/ml is associated with a human subject identified as a glatiramer acetate responder.

In an embodiment, the biomarker is IFN-γ concentration. In a further embodiment, an IFN-γ concentration greater than or equal to 6000 pg/ml is associated with a human subject identified as a glatiramer acetate responder.

In an embodiment, the biomarker is IL-2 concentration. In a further embodiment, an IL-2 concentration greater than or equal to 30000 pg/ml is associated with a human subject identified as a glatiramer acetate responder.

In an embodiment, the human subject is a naive patient.

In an embodiment, the human subject has been previously administered a multiple sclerosis drug other than glatiramer acetate. In a further embodiment, the previously administered multiple sclerosis drug is selected from Interferon, Mitoxantrone and Natalizumab.

This invention also provides a method for treating a human subject afflicted with multiple sclerosis or a single clinical attack consistent with multiple sclerosis comprising the steps of administering to the human subject a therapeutic amount of a pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier, determining whether the human subject is a glatiramer acetate responder by evaluating a biomarker selected from the group consisting of IL-17 concentration, TNF-α concentration, IL-2 concentration and IFN-γ concentration, or a combination thereof, in the blood of the human subject, and continuing administration of the pharmaceutical composition if the human subject is identified as a glatiramer acetate responder, or modifying the administration of the pharmaceutical composition to the human subject if the human subject is not identified as a glatiramer acetate responder.

In an embodiment, administering to the human subject a therapeutic amount of a pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier comprises administering to the human subject three subcutaneous injections of the pharmaceutical composition over a period of seven days with at least one day between every subcutaneous injection.

In an embodiment, the pharmaceutical composition is a unit dose of a 0.5 ml aqeuous solution comprising 20 mg of glatiramer acetate.

In an embodiment, the IL-17 concentration, INF-α concentration, IL-2 concentration and IFN-γ concentration, or the combination thereof is a PBMC supernatant concentration.

In an embodiment, the IL-17 concentration, INF-α concentration, IL-2 concentration and IFN-γ concentration, or the combination thereof is observed at pretreatment.

In an embodiment, the IL-17 concentration, INF-α concentration, IL-2 concentration and INF-γ concentration, or the combination thereof is observed at 2 months after the first administration of glatiramer acetate.

In an embodiment, if the human subject is identified as a glatiramer acetate responder, the human subject is thereafter administered the pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier as monotherapy.

In an embodiment, if the human subject is identified as a glatiramer acetate responder, the human subject is thereafter administered a pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier, and the human subject is also thereafter administered another multiple sclerosis drug which is not glatiramer acetate. In a further embodiment, the multiple sclerosis drug is selected from Interferon, Mitoxantrone and Natalizumab.

In an embodiment, if the human subject is not identified as a glatiramer acetate responder, the human subject is thereafter administered a multiple sclerosis drug which is not glatiramer acetate.

In an embodiment, if the human subject is not identified as a glatiramer acetate responder, the human subject is thereafter administered a multiple sclerosis drug which is not glatiramer acetate, and the human subject is not thereafter administered glatiramer acetate.

In an embodiment, the multiple sclerosis drug is selected from Interferon, Mitoxantrone and Natalizumab.

In an embodiment, the biomarker is IL-17 concentration.

In an embodiment, the biomarker is IL-17(A) concentration. In a further embodiment, an IL-17 concentration or an IL-17(A) concentration greater than or equal to 120 pg/ml is associated with a human subject identified as a glatiramer acetate responder.

In an embodiment, the biomarker is TNF-α concentration. In a further embodiment, a TNF-α concentration greater than or equal to 20000 pg/ml is associated with a human subject identified as a glatiramer acetate responder.

In an embodiment, the biomarker is IFN-γ concentration. In a further embodiment, an IFN-γ concentration greater than or equal to 6000 pg/ml is associated with a human subject identified as a glatiramer acetate responder.

In an embodiment, the biomarker is IL-2 concentration. In a further embodiment, an IL-2 concentration greater than or equal to 30000 pg/ml is associated with a human subject identified as a glatiramer acetate responder.

In an embodiment, the human subject is a naive patient.

In an embodiment, the human subject has been previously administered a multiple sclerosis drug other than glatiramer acetate. In a further embodiment, the previously administered multiple sclerosis drug is selected from Interferon, Mitoxantrone and Natalizumab.

DEFINITIONS Forms of Multiple Sclerosis

There are five distinct disease stages and/or types of MS:

-   -   1) benign multiple sclerosis;     -   2) relapsing-remitting multiple sclerosis (RRMS);     -   3) secondary progressive multiple sclerosis (SPMS);     -   4) progressive relapsing multiple sclerosis (PRMS); and     -   5) primary progressive multiple sclerosis (PPMS).

Benign multiple sclerosis is a retrospective diagnosis which is characterized by 1-2 exacerbations with complete recovery, no lasting disability and no disease progression for 10-15 years after the initial onset. Benign multiple sclerosis may, however, progress into other forms of multiple sclerosis.

Patients suffering from RRMS experience sporadic exacerbations or relapses, as well as periods of remission. Lesions and evidence of axonal loss may or may not be visible on MRI for patients with RRMS.

SPMS may evolve from RRMS. Patients afflicted with SPMS have relapses, a diminishing degree of recovery during remissions, less frequent remissions and more pronounced neurological deficits than RRMS patients. Enlarged ventricles, which are markers for atrophy of the corpus callosum, midline center and spinal cord, are visible on MRI of patients with SPMS.

PPMS is characterized by a steady progression of increasing neurological deficits without distinct attacks or remissions.

Cerebral lesions, diffuse spinal cord damage and evidence of axonal loss are evident on the MRI of patients with PPMS. PPMS has periods of acute exacerbations while proceeding along a course of increasing neurological deficits without remissions. Lesions are evident on MRI of patients suffering from PRMS. (28)

A clinically isolated syndrome (CIS) is a single monosymptomatic attack compatible with MS, such as optic neuritis, brain stem symptoms, and partial myelitis. Patients with CIS that experience a second clinical attack are generally considered to have clinically definite multiple sclerosis (CDMS). Over 80 percent of patients with a CIS and MRI lesions go on to develop MS, while approximately 20 percent have a self-limited process.(29,30) Patients who experience a single clinical attack consistent with MS may have at least one lesion consistent with multiple sclerosis prior to the development of clinically definite multiple sclerosis.

Multiple sclerosis may present with optic neuritis, blurring of vision, diplopia, involuntary rapid eye movement, blindness, loss of balance, tremors, ataxia, vertigo, clumsiness of a limb, lack of co-ordination, weakness of one or more extremity, altered muscle tone, muscle stiffness, spasms, tingling, paraesthesia, burning sensations, muscle pains, facial pain, trigeminal neuralgia, stabbing sharp pains, burning tingling pain, slowing of speech, slurring of words, changes in rhythm of speech, dysphagia, fatigue, bladder problems (including urgency, frequency, incomplete emptying and incontinence), bowel problems (including constipation and loss of bowel control), impotence, diminished sexual arousal, loss of sensation, sensitivity to heat, loss of short term memory, loss of concentration, or loss of judgment or reasoning.

Relapsing Form of Multiple Sclerosis:

The term relapsing MS includes:

-   -   1) patients with RRMS;     -   2) patients with SPMS and superimposed relapses; and     -   3) patients with CIS who show lesion dissemination on subsequent         MRI scans according to McDonald's criteria.

As used herein, relapsing forms of multiple sclerosis include: Relapsing-remitting multiple sclerosis (RRMS), characterized by unpredictable acute episodes of neurological dysfunction (relapses), followed by variable recovery and periods of clinical stability;

Secondary Progressive MS (SPMS), wherein patients having RRMS develop sustained deterioration with or without relapses superimposed; and

Primary progressive-relapsing multiple sclerosis (PPRMS) or progressive-relapsing multiple sclerosis (PRMS), an uncommon form wherein patients developing a progressive deterioration from the beginning can also develop relapses later on.

Kurtzke Expanded Disability Status Scale (EDSS):

The Kurtzke Expanded Disability Status Scale (EDSS) is a method of quantifying disability in multiple sclerosis. The EDSS replaced the previous Disability Status Scales which used to bunch people with MS in the lower brackets. The EDSS quantifies disability in eight Functional Systems (FS) and allows neurologists to assign a Functional System Score (FSS) in each of these. The Functional Systems are: pyramidal, cerebellar, brainstem, sensory, bowel and bladder, visual & cerebral (according to www.mult-sclerosis.org/expandeddisabil itystatusscale).

Clinical Relapse:

A clinical relapse, which may also be used herein as “relapse,” “confirmed relapse,” or “clinically defined relapse,” is defined as the appearance of one or more new neurological abnormalities or the reappearance of one or more previously observed neurological abnormalities.

This change in clinical state must last at least 48 hours and be immediately preceded by a relatively stable or improving neurological state of at least 30 days. This criterion is different from the clinical definition of exacerbation “at least 24 hours duration of symptoms,” (31) as detailed in the section “relapse evalution.”

An event is counted as a relapse only when the subject's symptoms are accompanied by observed objective neurological changes, consistent with:

a) an increase of at least 1.00 in the EDSS score or one grade in the score of two or more of the seven FS (32); or, b) two grades in the score of one of FS as compared to the previous evaluation.

The subject must not be undergoing any acute metabolic changes such as fever or other medical abnormality. A change in bowel/bladder function or in cognitive function must not be entirely responsible for the changes in EDSS or FS scores.

As used herein, “in the blood of the subject” is represented by “serum” and also the “supernatant” of PBMCs derived from the subject's blood.

As used herein, the “supernatant” refers to supernatants collected from Peripheral blood mononuclear cells (PBMCs) purified from subject blood samples, and stimulated as described in the methods hereinbelow. The stimulation may be performed in either freshly isolated PBMCs or in cryopreserved cells after thawing.

As used herein, “concentration observed at” a certain time-point refers to a concentration measured in the supernatant of PBMC derived from the subject's blood at that certain time point. The concentration may be measured in freshly isolated cells or in cryopreserved cells after thawing.

As used herein, “pretreatment” refers to any time point after diagnosis with MS or CIS and before beginning of treatment with a composition comprising GA.

As used herein, a “multiple sclerosis drug” is a drug or an agent intended to treat clinically defined MS, CIS, any form of neurodegenerative or demyelinating diseases, or symptoms of any of the above mentioned diseases. “Multiple sclerosis drugs” may include but are not limited to antibodies, immunosuppressants, anti-inflammatory agents, immunomodulators, cytokines, cytotoxic agents and steroids and may include approved drugs, drugs in clinical trial, or alternative treatments, intended to treat clinically defined MS, CIS or any form of neurodegenerative or demyelinating diseases. “Multiple sclerosis drugs” include but are not limited to Interferon and its derivatives (including BETASERON®, AVONEX® and REBIF®), Mitoxantrone and Natalizumab. Agents approved or in-trial for the treatment of other autoimmune diseases, but used in a MS or CIS patient to treat MS or CIS are also defined as multiple sclerosis drugs.

As used herein, a “naïve patient” is a subject that has not been treated with any multiple sclerosis drugs as defined in the former paragraph.

EXPERIMENTAL DETAILS Example Evaluating Cytokine Levels in Patients Classified as Responders or Non-Responders to GA Methods Subjects and Cells:

Relapsing-remitting multiple sclerosis patients (n=12) were treated with either 20 mg GA or 40 mg GA daily in the Teva FORTE clinical trial(www.medicalnewstoday.com/articles/48863.php). Whole blood was taken from patients at three time points including baseline (baseline, month 2 and month 6). Peripheral blood mononuclear cells (PBMCs) were cryopreserved at baseline, month 2 and month 6.

The incidence of clinical relapses, and the expanded disability status scale score (EDSS) after 12 months treatment were used to define patients as responders (no clinical relapse during the test period) or non-responders (1 or more clinical relapses as defined hereinbelow). Several patients were withdrawn from drug within the treatment year due to adverse responses and were not included in this analysis.

Multiplex Cytokine Assay

Blood was drawn from patients at baseline, 2 months and 6 months. Peripheral Blood Mononuclear Cells (PBMCs) were purified from the blood using a Ficoll-Hypaque gradient and cryopreserved. Cryopreserved PBMCs from each time point were thawed, rested overnight in AIM V medium supplemented with 5% human serum, and stimulated with PMA (1 mg/ml; SIGMA) and ionomycin (5 mg/ml; SIGMA) for 6 hours (40,000 PBMCs in 200 microliters final volume). Supernatants were removed from stimulated and unstimulated cells and stored at −20oC until assay with a human 27-plex kit (Bio-Rad Laboratories, Hercules, Calif.). Data was acquired using a Bio-Plex Array Reader and analyzed with Bio-Plex Manager 4 software (Bio-Rad). Graphs were drawn using Prism software (GraphPad Software, Inc.). We performed both 2-plex (IL-17 and IFNy) and 27 multiplex (including 27 human cytokines) were used in this comprehensive cytokine analysis. The multiplex data from these two assays were individualized on a per patient basis and presented in FIG. 2.

Relapse Evaluation

A clinical relapse was defined as the appearance of one or more new neurological abnormalities or the reappearance of one or more previously observed neurological abnormalities.

This change in clinical state lasted at least 48 hours and was immediately preceded by a relatively stable or improving neurological state of at least 30 days. The criterion used in the study was different from the clinical definition of exacerbation “at least 24 hours duration of symptoms”. (31) Since “in study” exacerbation definition must be supported by an objective neurological evaluation (see next paragraph), a neurological deficit must sustain long enough to eliminate pseudo exacerbations.

An event was counted as a relapse only when the subject's symptoms were accompanied by observed objective neurological changes, consistent with:

a) an increase of at least 1.00 in the EDSS score or one grade in the score of two or more of the seven FS (32); or, b) two grades in the score of one of FS as compared to the previous evaluation.

The subject was not undergoing any acute metabolic changes such as fever or other medical abnormality. A change in bowel/bladder function or in cognitive function was not entirely responsible for the changes in EDSS or FS scores.

Subject Evaluation by the Examining Neurologist

A complete neurological assessment was performed at months −1 (screening), 0 (baseline), 3, 6, 9, 12 (end of double-blind phase), 18 and 24 (termination/early discontinuation).

Relapse Determination by the Treating Neurologist

The decision as to whether the neurological change was considered a confirmed relapse was made by the Treating Physician, based on EDSS/FS actual (not converted) scores assessed by the Examining Neurologist.

Follow-up visits to monitor the course of the relapse were made at the Treating Physician's discretion, in addition to the assessment at the next scheduled visit, but the neurological assessments were performed by the Examining Neurologist.

Relapse Evaluation Procedures

Subjects were instructed to telephone their study site within 48 hours should any symptoms suggestive of a relapse appear.

The Examining Neurologist evaluated the subject within 7 days of symptoms onset, conditional upon a symptomatic period of ≧48 hours. The Treating Neurologist/Physician evaluated the subject once any symptom suggestive of a relapse occurred.

In case of a suggestive relapse during a scheduled or unscheduled visit, the Treating Neurologist/Physician referred the subject to the Examining eurologist/Physician.

Results

These findings demonstrate increased levels of IL-17(A), TNF-alpha, IL-2 and IFN-gamma, at the baseline and 2 months time points, in those individuals who were without clinically defined relapses in the one year trial period post initiation of treatment. To the contrary, we observed substantially lower levels of these same pro-inflammatory cytokines in those who had clinically defined relapses during the trial period (see FIG. 1 and FIG. 2).

Discussion

For this study, PBMC were obtained at baseline, 2 months and 6 months post initiation of treatment with glatiramer acetate. The findings demonstrate increased levels of IL-17(A), IL-2, TNFα, and IFNγ at baseline and 2 months after the beginning of treatment with GA, in those individuals who were without clinically defined relapses in the one year trial period post initiation of treatment. To the contrary, substantially lower levels of these same pro-inflammatory cytokines were found in those who had clinically defined relapses during the trial period as shown in FIG. 1.

Ex vivo assays have been used to monitor the immunological effects of GA in GA-treated MS patients. For example, Hohlfeld et al. reported: (1) a significant reduction of GA-induced PBMC proliferation; (2) a positive IL-4 ELISPOT response mediated predominantly by CD4 cells after stimulation with GA; and (3) an elevated IFN-gamma response partially mediated by CD8 cells after stimulation with high GA concentrations, in GA-treated vs. untreated patients (33).

In the present study, a simple ex vivo assay was used to measure cytokine concentration in the supernatant of PBMCs derived from the blood of RRMS patients. The data suggest that specific cytokine patterns may be associated with the identification of those who will respond to therapy with glatiramer acetate. The trend that is seen may be suggestive of cytokine patterns that could be readily measured and assist in determining GA responsiveness before GA treatment, and at an early time-point after the beginning of GA administration.

REFERENCES

-   1. Noseworthy J H, Lucchinetti C, Rodriguez M, Weinshenker BG.     Multiple sclerosis. N Engl J Med 2000; 343:938-52. -   2. Guideline on clinical investigation of medicinal products for the     treatment of multiple sclerosis EMEA, London 16 Sep. 2006. -   3. Bjartmar C, Fox R J. Pathological mechanisms and disease     progression of multiple sclerosis: therapeutic implications. Drugs     of Today 2002; 38:17-29. -   4. Fleming J O. Diagnosis and management of multiple sclerosis. 1st     ed. New York: Professional communications, Inc., 2002. -   5. Anderson D W, Ellenberg J H, Leventhal C M et al. Revised     estimate of the prevalence of multiple sclerosis in the United     States. Ann Neurol 1992; 31:333-36. -   6. Compston A, Lassmann H, McDonald I. The story of multiple     sclerosis. In: Compston A, Confavreux C, Lassman H, Mcdonald I,     Miller D, Noseworthy J H, Smith K, Wekerle H, editors. McAlpine's     Multiple Sclerosis. London: Churchill Livingstone; 2006. p. 3-68. -   7. Revel M., Pharmacol. Ther., 100(1):49-62 (2003). -   8. Martinelli B F, Rovaris M, Johnson K P, Miller A, Wolinsky J S,     Ladkani D, Shifroni G, Comi G, Filippi M. Effects of glatiramer     acetate on relapse rate and accumulated disability in multiple     sclerosis: meta-analysis of three double-blind, randomized,     placebo-controlled clinical trials. Mult Scler. 2003 August;     9(4):349-55. -   9. Mikol D D, Barkhof F, Chang P, Coyle P K, Jeffery D R, Schwid S     R, Stubinski B, Uitdehaag B M; REGARD study group. Lancet Neurol.     2008 October; 7(10):903-14. Epub 2008 Sep. 11. -   10. BECOME TRIAL, Presented at the 23rd Congress of the European     Committee for Treatment and Research in Multiple Sclerosis (ECTRIMS)     in Prague, Czech Republic. -   11. Comi G, Filippi M and Wolinsky J S. European/Canadian     multi-center, double-blind randomized, placebo controlled study of     the effects of glatiramer acetate on magnetic resonance     imaging-measured disease activity and burden in patients with     relapsing-remitting multiple sclerosis. Ann Neurol 2001;     (49):290-297. -   12. Fridkis H M, Aharoni R, Teitelbaum D, Arnon R, Sela M,     Strominger J L. Binding of random copolymers of three amino acids to     class II MHC molecules. Int. Immunol. 1999 May; 11(5):635-41. -   13. Dhib-Jalbut S S, Zhan M, Johnson K P, Martin R. Glatiramer     acetate reactive blood mononuclear cells respond to myelin antigens     with a Th-2 biased phenotype. J Neuroimmunology 2003; 140:163-171. -   14. Chen M, Gran B, Costello K, Johnson K P, Martin R,     Dhib-Jalbut S. Glatiramer acetate induces a Th-2 biased response and     cross-reactivity with myelin basic protein in patients with MS.     Multiple Sclerosis 2001; 7:209-219. -   15. Weber M S, Prod'homme T, Youssef S, Dunn S E, Rundle C D, Lee L,     Patarroyo J C, Stüve O, Sobel R A, Steinman L, Zamvil S S. Type II     monocytes modulate T cell-mediated central nervous sytem autoimmune     disease. Nat Med (2007) 13:935-943. -   16. Aharoni R, Kayhan B, Eilam R, Sela M, and Arnon R. Glatiramer     acetate-specific T cells in the brain express T helper ⅔ cytokines     and brain-derived neurotrophic factor in situ. PNAS August 2003;     100(24):14157-62. -   17. Sarchielli P, Zaffaroni M, Floridi A, Greco L, Candeliere A,     Mattioni A, Tenaglia S, Di Filippo M, Calabresi P. Production of     brain-derived neurotrophic factor by mononuclear cells of patients     with multiple sclerosis treated with glatiramer acetate,     interferon-beta 1a, and high doses of immunoglobulins. Mult Scler     2007 April; 13(3):313-31. Epub 2007 Jan. 29. -   18. Bornstein, M B, Miller, A, Slagle, S, et al. A pilot trial of     Cop 1 in exacerbating remitting multiple sclerosis. New Eng J Med     1987; 317: 408-14. -   19. Comi, G, Fillippi, M, Wolinsky, J S, et al. European/Canadian     multicenter, double-blind, randomized, placebo-controlled study of     the effects of glatiramer acetate on magnetic resonance     imagine-measured disease activity and burden in patients with     relapsing multiple sclerosis. Ann Neurol 2001; 49: 290-7. -   20. Johnson, K P, Brooks, B R, Cohen, J A, et al. Extended use of     glatiramer acetate (Copaxone) is well tolerated and maintains its     clinical effect on multiple sclerosis relapse rate and degree of     disability. Neurology 1998; 50:701-8. -   21. Bornstein, M B, Miller, A, Slagle, S, et al. A     placebo-controlled, double-blind, randomized, two-center, pilot     trial of Cop-1 in chronic progressive multiple sclerosis. Neurology     1991; 41: 533-39. -   22. Wolinsky, J S, Narayana, P A, O'Conner, P, et al. -   Glatiramer acetate in primary progressive multiple sclerosis:     Results of a multinational, multicenter, double-blind,     placebo-controlled trial. Ann Neurol 2007; 61:14-24. -   23. Comi G, Filippi M, Treatment with glatiramer acetate delays     conversion to clinically definite multiple sclerosis (CDMS) in     patients with clinically isolated syndromes (CIS). Neurology 2008;     71(2): 153. -   24. Tselis, A, Khan, O, Lisak, R P, Glatiramer acetate in the     treatment of multiple sclerosis. Neuropsychiatric Dis Treat 2007;     3(2):259-67. -   25. Wolinsky, J S, The use of glatiramer acetate in the treatment of     multiple sclerosis. Adv Neurol 2006; 273-92. -   26. Comi G, Cohen J A, Filippi M, Results from a phase III,     one-year, randomized, double-blind, parallel-group, dose-comparison     study with glatiramer acetate in relapsing-remitting multiple     sclerosis. Mult Scler 2008; 14(suppl 1):S299. -   27. Comi G, Filippi M. Presented at: 60th Annual Meeting of the     American Academy of Neurology: April 12-19; Chicago, Ill. Abstract     LBS.003. -   28. Johnson D, Hafler D A, Fallis R J, Lees M B, Brady R O, Quarles     R H, Weiner H L., “Cell-mediated immunity to myelin-associated     glycoprotein, proteolipid protein, and myelin basic protein in     multiple sclerosis.”, J Neuroimmunol. 1986 November; 13 (1):99-108. -   29. Brex P A et al., “A longitudinal study of abnormalities on MRI     and disability from multiple sclerosis”, N Engl J Med 2002 Jan. 17,     346(3):158-64. -   30. Frohman E M et al., “The utility of MRI in suspected MS: report     of the Therapeutics and Technology Assessment Subcommittee of the     American Academy of Neurology”, Neurology, 2003, Sep. 9, 61(5):     602-11. -   31. Poser C M. et al. New diagnostic criteria for multiple     sclerosis: Guidelines for research protocols. Ann. Neurol., 13(3):     227-31, 1983 -   32. Neurostatus, slightly modified from J. F. Kurtzke Neurology     1983:33, 1444-52; L. Kappos, Dept. of Neurology, University     Hospital, CH-4031/Basel, Switzerland. -   33. Farina C, Then Bergh F, Albrecht H, Meinl E, Yassouridis A,     Neuhaus O, Hohlfeld R. Treatment of multiple sclerosis with Copaxone     (COP): Elispot assay detects COP-induced interleukin-4 and     interferon-gamma response in blood cells. Brain. 2001 April; 124(Pt     4):705-19. 

1. A method for treating a human subject afflicted with multiple sclerosis or a single clinical attack consistent with multiple sclerosis with a pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier, comprising the steps of: a) determining whether the human subject is a glatiramer acetate responder by evaluating TNF-α concentration in the blood of the human subject; and b) administering the pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier to the human subject only if the human subject is identified as a glatiramer acetate responder.
 2. The method of claim 1, wherein administering the pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier comprises administering to the human subject three subcutaneous injections of the pharmaceutical composition over a period of seven days with at least one day between every subcutaneous injection.
 3. The method of claim 1, wherein the pharmaceutical composition is a unit dose of a 0.5 ml aqueous solution comprising 20 mg of glatiramer acetate.
 4. A method of predicting clinical responsiveness to glatiramer acetate therapy in a human subject afflicted with multiple sclerosis or a single clinical attack consistent with multiple sclerosis, the method comprising evaluating TNF-α concentration in the blood of the human subject, to thereby predict clinical responsiveness to glatiramer acetate.
 5. The method of claim 4, wherein the glatiramer acetate therapy comprises administering to the human subject three subcutaneous injections of a pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier over a period of seven days with at least one day between every subcutaneous injection.
 6. The method of claim 5, wherein the pharmaceutical composition is a unit dose of a 0.5 ml aqueous solution comprising 20 mg of glatiramer acetate.
 7. A method for treating a human subject afflicted with multiple sclerosis or a single clinical attack consistent with multiple sclerosis comprising the steps of: a) administering to the human subject a therapeutic amount of a pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier; b) determining whether the human subject is a glatiramer acetate responder by evaluating TNF-α concentration in the blood of the human subject; and c) continuing administration of the pharmaceutical composition if the human subject is identified as a glatiramer acetate responder, or modifying the administration of the pharmaceutical composition to the human subject if the human subject is not identified as a glatiramer acetate responder.
 8. The method of claim 7, wherein administering to the human subject a therapeutic amount of a pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier comprises administering to the human subject three subcutaneous injections of the pharmaceutical composition over a period of seven days with at least one day between every subcutaneous injection.
 9. The method of claim 7, wherein the pharmaceutical composition is a unit dose of a 0.5 ml aqueous solution comprising 20 mg of glatiramer acetate.
 10. The method of claim 1, wherein the TNF-α concentration, is PBMC supernatant concentration.
 11. The method of claim 1, wherein the TNF-α concentration is observed at pretreatment.
 12. The method of claim 1, wherein the TNF-α concentration is observed at 2 months after the first administration of glatiramer acetate.
 13. The method of claim 1, wherein if the human subject is identified as a glatiramer acetate responder, the human subject is thereafter administered the pharmaceutical composition comprising glatiramer acetate and a pharmaceutically acceptable carrier as monotherapy.
 14. The method of claim 1, wherein if the human subject is not identified as a glatiramer acetate responder, the human subject is thereafter administered a multiple sclerosis drug which is not glatiramer acetate. 15-20. (canceled)
 21. The method of claim 1, wherein a TNF-α concentration greater than or equal to 20000 pg/ml is associated with a human subject identified as a glatiramer acetate responder. 22.-23. (canceled)
 24. The method of claim 1, wherein the human subject is a naive patient.
 25. The method of claim 1, wherein the human subject has been previously administered a multiple sclerosis drug other than glatiramer acetate.
 26. The method of claim 1, wherein step (a) further comprises evaluating a biomarker selected from the group consisting of IL-2 concentration and IFN-γ concentration, or a combination thereof, in the blood of the human subject.
 27. The method of claim 4, further comprising evaluating a biomarker selected from the group consisting of IL-2 concentration and IFN-γ concentration, or a combination thereof, in the blood of the human subject.
 28. The method of claim 7, wherein step (b) further comprises evaluating a biomarker selected from the group consisting of IL-2 concentration and IFN-γ concentration, or a combination thereof, in the blood of the human subject. 